The present invention relates to lysophospholipases (LPL), methods of using and producing them, as well as nucleic acid sequences encoding them.
Lysophospholipases (EC 3.1.1.5) are enzymes that can hydrolyze 2-lysophospholids to release fatty acid. They are known to be useful, e.g., for improving the filterability of an aqueous solution containing a starch hydrolysate, particularly a wheat starch hydrolysate (EP 219.269).
N. Masuda et al., Eur. J. Biochem., 202, 783-787 (1991) describe an LPL from Penicillium notatum as a glycoprotein having a molecular mass of 95 kDa and a published amino acid sequence of 603 amino acid residues. WO 98/31790 and EP 808,903 describe LPL from Aspergillus foetidus and Aspergillus niger, each having a molecular mass of 36 kDa and an amino acid sequence of 270 amino acids.
JP-A 10-155493 describes a phospholipase A1 from Aspergillus oryzae. The mature protein has 269 amino acids.
The Inventors have isolated lysophospholipases from Aspergillus (A. niger and A. oryzae) having molecular masses of about 68 kDa and amino acid sequences of 600-604 amino acid residues. The novel lysophospholipases have only a limited homology to known amino acid sequences. The inventors also isolated genes encoding the novel enzymes and cloned them into E. coil strains.
Accordingly, the invention provides a lysophospholipase which may be a polypeptide having an amino acid sequence as the mature peptide shown in one of the following or which can be obtained therefrom by substitution, deletion, and/or insertion of one or more amino acids, particularly by deletion of 25-35 amino acids at the C-terminal:
SEQ ID NO: 2 (hereinafter denoted A. niger LLPL-1),
SEQ ID NO: 4 (hereinafter denoted A. niger LLPL-2),
SEQ ID NO: 6 (hereinafter denoted A. oryzae LLPL-1), or
SEQ ID NO: 8 (hereinafter denoted A. oryzae LLPL-2).
Further, the lysophospholipase of the invention may be a polypeptide encoded by the lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli deposit number DSM 13003, DSM 13004, DSM 13082 or DSM 13083.
The lysophospholipase may also be an analogue of the polypeptide defined above which:
i) has at least 70% homology with said polypeptide,
ii) is immunologically reactive with an antibody raised against said polypeptide in purified form,
iii) is an allelic variant of said polypeptide,
Finally, the phospholipase of the invention may be a polypeptide which is encoded by a nucleic acid sequence which hybrdizes under high stringency conditions with one of the following sequences or its complementary strand or a subsequence thereof of at least 100 nucleotides:
nucleotides 109-1920 of SEQ ID NO: 1 (encoding A. niger LLPL-1),
nucleotides 115-1914 of SEQ ID NO: 3 (encoding A. niger LLPL-2),
nucleotides 70-1881 of SEQ ID NO: 5 (encoding A. oryzae LLPL-1), or
nucleotides 193-2001 of SEQ ID NO: 7 (encoding A. oryzae LLPL-2).
The nucleic acid sequence of the invention may comprise a nucleic acid sequence which encodes any of the lysophospholipases described above, or it may en-code a lysophospholipase and comprise:
a) the lysophospholipase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli DSM 13003, DSM 13004, DSM 13082 or DSM 13083 (encoding A. niger LLPL-1, A. niger LLPL-2, A. oryzae LLPL-1 and A. oryzae LLPL-2, respectively),
b) the DNA sequence shown in SEQ ID NO: 1, 3, 5 or 7 (encoding A. niger LLPL-1, A. niger LLPL-2, A. oryzae LLPL-1 and A. oryzae LLPL-2, respectively), or
c) an analogue of the DNA sequence defined in a) or b) which
i) has at least 70% homology with said DNA sequence, or
ii) hybridizes at high stringency with said DNA sequence, its complementary strand or a subsequence thereof.
Other aspects of the invention provide a recombinant expression vector comprising the DNA sequence, and a cell transformed with the DNA sequence or the recombinant expression vector.
A comparison with full-length prior-art sequences shows that the mature amino acid sequences of the invention have 60-69% homology with LPL from Penicillium notatum (described above), and the corresponding DNA sequences of the invention show 63-68% homology with that of P. notatum LPL.
A comparison with published partial sequences shows that an expressed sequence tag (EST) from Aspergillus nidulans (GenBank M965865) of 155 amino acid residues can be aligned with the mature A. oryzae LLPL-2 of the invention (604 amino acids) with a homology of 79%.
Lysophospholipases of the invention may be derived from strains of Aspergillus, particularly strains of A. niger and A. oryzae, using probes designed on the basis of the DNA sequences in this specification.
Strains of Escherichia coli containing genes encoding lysophospholipase were deposited by the inventors under the terms of the Budapest Treaty with the DSMZxe2x80x94Deutsche Sammlung von Microorganismen und Zelikulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig DE as follows:
The lysophospholipase may be derived from the mature peptide shown in SEQ ID NOS: 2, 4, 6 or 8 by deletion at the C-terminal to remove the xcfx89 site residue; while preserving the lysophospholipase activity. The xcfx89 site residue is described in Yoda et al. Biosci. Biotechnol. Biochem. 64, 142-148, 2000, e.g. S577 of SEQ ID NO: 4. Thus, the C-terminal deletion may particularly consist of 25-35 amino acid residues.
A lysophospholipase with a C-terminal deletion may particularly be produced by expression in a strain of A. oryzae. 
The lysophospholipase of the invention is able to hydrolyze fatty acyl groups in lysophospholipid such as lyso-lecithin (Enzyme Nomendature EC 3.1.1.5). It may also be able to release fatty acids from intact phospholipid (e.g. lecithin).
The expression vector of the invention typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a selectable marker, a transcription terminator, a repressor gene or various activator genes. The vector may be an autonomously replicating vector, or it may be integrated into the host cell genome.
The lysophospholipase of the invention may be produced by transforming a suitable host cell with a DNA sequence encoding the phospholipase, cultivating the transformed organism under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
The host organism is preferably a eukaryotic cell, in particular a fungal cell, such as a yeast cell or a filamentous fungal cell, such as a strain of Aspergillus, Fusarium, Trichoderma or Saccharomyces, particularly A. niger, A. oryzae, F. grominearm, F. sambucinum, F. cerealis or S. cerevisiae, e.g. a glucoamylase-producing strain of A. niger such as those described in U.S. Pat. No. 3,677,902 or a mutant thereof. The production of the lysophospholipase in such host organisms may be done by the general methods described in EP 238,023 (Novo Nordisk), WO 96/00787 (Novo Nordisk) or EP 244,234 (Alko).
The hybridization is used to indicate that a given DNA sequence is analogous to a nucleotide probe corresponding to a DNA sequence of the invention. The hybridization conditions are described in detail below.
Suitable conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5xc3x97SSC (standard saline citrate) for 10 min, and prehybridization of the filter in a solution of 5xc3x97SSC (Sambrook et al. 1989), 5xc3x97Denhardt""s solution (Sambrook et al. 1989), 0.5% SDS and 100 xcexcg/ml of denatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed by hybridization in the same solution containing a random-primed (Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity  greater than 1xc3x97109 cpm/xcexcg) probe for 12 hours at approx. 45xc2x0 C. The filter is then washed two times for 30 minutes in 2xc3x97SSC, 0.5% SDS at a temperature of at least 55xc2x0 C., more preferably at least 60xc2x0 C., more preferably at least 65xc2x0 C., even more preferably at least 70xc2x0 C., especially at least 75xc2x0 C.
Molecules to which the oligonucleotide probe hybridizes under these conditions are detected using a x-ray film.
The lysophospholipase and the nucleotide sequence of the invention preferably have homologies to the disclosed sequences of at least 80%, particularly at least 90% or at least 95%, e.g. at least 98%.
For purposes of the present invention, alignments of sequences and calculation of homology scores were done using a full Smith-Waterman alignment, useful for both protein and DNA alignments. The default scoring matrices BLOSUM50 and the identity matrix are used for protein and DNA alignments respectively. The penalty for the first residue in a gap is xe2x88x9212 for proteins and xe2x88x9216 for DNA, while the penalty for additional residues in a gap is xe2x88x922 for proteins and xe2x88x924 for DNA. Alignment is from the FASTA package version v20u6 (W. R. Pearson and D. J. Lipman (1988), xe2x80x9cImproved Tools for Biological Sequence Analysisxe2x80x9d, PNAS 85:2444-2448, and W. R. Pearson (1990) xe2x80x9cRapid and Sensitive Sequence Comparison with FASTP and, FASTAxe2x80x9d, Methods in Enzymology, 183:63-98). Multiple alignments of protein sequences were done using xe2x80x9cClustalWxe2x80x9d (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680). Multiple alignment of DNA sequences are done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence.
Lysophospholipase activity is measured using egg yolk L-xcex1-lysolecithin as the substrate with a NEFA C assay kit.
20 xcexcl of sample is mixed with 100 xcexcl of 20 mM sodium acetate buffer (pH 4.5) and 100 xcexcl of 1% L-xcex1-lysolecithin solution, and incubated at 55xc2x0 C. for 20 min. After 20 min, the reaction mixture is transferred to the tube containing 30 xcexcl of Solution A in NEFA kit preheated at 37xc2x0 C. After 10 min incubation at 37xc2x0 C., 600 xcexcl of Solution B in NEFA kit is added to the reaction mixture and incubated at 37xc2x0 C. for 10 min. Activity is measured at 555 nm on a spectrophotometer. One unit of lysophospholipase activity (1 LLU) is defined as the amount of enzyme that can increase the A550 of 0.01 per minute at 55xc2x0 C.
The lysophospholipase of the invention can be used in any application where it is desired to hydrolyze the fatty acyl group(s) of a phospholipid or lyso-phospholipid, such as lecithin or lyso-lecithin.
As an example, the lysophospholipase of the invention can be used in the preparation of dough, bread and cakes, e.g. to improve the elasticity of the bread or cake. Thus, the lysophospholipase can be used in a process for making bread, comprising adding the lysophospholipase to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with U.S. Pat. No. 4,567,046 (Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.) or EP 426211 (Unilever).
The lysophospholipase of the invention can also be used to improve the filter-ability of an aqueous solution or slurry of carbohydrate origin by treating it with the lysophospholipase. This is particularly applicable to a solution or slurry containing a starch hydrolysate, especially a wheat starch hydrolysate since this tends to be difficult to filter and to give cloudy filtrates. The lysophospholipase may advantageously be used together with a beta-glucanase and/or a xylanase, e.g. as described in EP 219,269 (CPC International).
The lysophospholipase of the invention can be used in a process for reducing the content of phospholipid in an edible oil, comprising treating the oil with the lysophospholipase so as to hydrolyze a major part of the phospholipid, and separating an aqueous phase containing the hydrolyzed phospholipid from the oil. This process is applicable to the purification of any edible oil which contains phospholipid, e.g. vegetable oil such as soy bean oil, rape seed oil and sunflower oil. The process can be conducted according to principles known in the art, e.g. in analogy with U.S. Pat. No. 5,264,367 (Metaligesellschaft, Rxc3x6hm); K. Dahike and H. Buchold, INFORM, 6 (12), 1284-91 (1995); H. Buchold, Fat Sci. Technol., 95 (8), 300-304 (1993); JP-A 2-153997 (Showa Sangyo); or EP 654,527 (Metallgesellschaft, Rxc3x6hm).